Cefotaxime Sodium Salt Antibiotic Protocol

The solvent used to prepare and dilute antibiotic Cefotaxime sodium salt (CTX, Sigma Aldrich) was the M9 compound medium to avoid the concentration of the nutrients decreasing in the prepared culture medium. The original antibiotic stock solution (25 mg/mL CTX solution) was prepared by dissolving 66.49 mg Cefotaxime sodium salt (potency is 940 μg/mg) into 2.5 mL M9 compound medium (Andrews, 2001 (link)). Afterward, the original CTX solution was filtered (VWR, 0.2 μm cellulose acetate membrane) and further diluted to various gradient concentrations. In the following, 300 μL solution with different concentrations was transferred in 500 μL Eppendorf tubes with correlated concentration labels. All the prepared antibiotic stock solutions were kept in the freezer at −20°C for a maximum of 2 months. To enable the expression of all TEM motifs under the control of lacI repressor, 50 μM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma Aldrich) was also added to the growth medium for each experiment (Gomes et al., 2020 (link)). The IPTG stock (5 mM) was prepared by dissolving 119.15 mg into a 100 mL M9 compound medium, followed by transferring 1 mL to 2 mL Eppendorf tubes and keeping it in the freezer at −20°C for a maximum of 2 months.